Polynucleotides for amplification and detection of sars-cov-2

ABSTRACT

Disclosed herein are primers and probes related to the detection of SARS-CoV-2 via nucleic acid amplification testing (NAAT), for example to amplify and determine the presence of SARS-CoV-2 in test samples and/or to diagnose Covid-19. Specifically, the present disclosure describes primers and probes that bind to the N gene, ORF1ab, or E gene of SARS-CoV-2 coronavirus for detection via loop mediated isothermal amplification (LAMP) and molecular beacon hybridization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/912,446, filed Jun. 25, 2020, which claims the benefit of U.S.Provisional Application No. 62/993,523, filed Mar. 23, 2020, and U.S.Provisional Application No. 63/009,803, filed Apr. 14, 2020, thecontents of which are each incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 20, 2021, isnamed TSM-055US1 D1_SL.txt and is 23,625 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the fields of molecular biology andnucleic acid chemistry. The invention provides methods and reagents fordetecting the SARS-CoV-2 virus, the causative agent for the diseasereferred to as COVID-19. The invention also relates to the fields ofmedical diagnostics and prognostics. In particular, the inventionrelates to polynucleotides and methods for amplifying and detectingcertain genes in the SARS-CoV-2 viral genome.

BACKGROUND

There is an urgent need for the development of a rapid, affordable,sample-in answer-out point of care (POC) diagnostic platform for thenovel coronavirus spreading across the globe. On 11 Mar. 2020, the WorldHealth Organization (WHO) declared COVID-19, which is caused by theSARS-CoV-2 virus, to be a pandemic based on 118,000 cases in 114countries and 4,291 deaths. Ten days later, the Johns HopkinsCoronavirus Resource Center reported 329,858 confirmed cases with 14,380deaths worldwide. Three months later, there are over 8.5 millionconfirmed cases worldwide and the death toll is nearly half a millionpeople with little indication that spread of the disease is slowing.Throughout this public health crisis, the inability to test members ofthe public for infection with SARS-CoV-2 has significantly hamperedefforts to contain the pandemic.

The compositions and methods disclosed herein provide primers and probesfor the detection of the SARS-CoV-2 RNA virus using loop-mediatedisothermal amplification.

SUMMARY OF THE INVENTION

The present invention encompasses, in some embodiments, a compositioncomprising a set of polynucleotides selected from the group consistingof Set-1 through Set-17. In some embodiments, the composition furthercomprises a probe. In some embodiments, the probe comprises a label. Insome embodiments, the probe is a labeled polynucleotide. In a preferredimplementation, the label is a fluorophore, which preferably iscovalently attached to a terminus of the polynucleotide. In aparticularly preferred embodiment, the probe or polynucleotide is amolecular beacon comprising a fluorophore, a quencher, and apolynucleotide. In one embodiment, the fluorophore is FAM and thequencher is Black Hole Quencher 1 (BHQ1; LGC Biosearch Technologies). Inan alternate implementation, the fluorophore is ATTO 565 or Alexa 594 orCy5 and the quencher is BHQ1 or BHQ2.

In some implementations, composition comprises a labeled polynucleotidecomprising a sequence selected from the group consisting of nucleotides5-27 of SEQ ID NO.: 55, nucleotides 7-25 of SEQ ID NO.: 56, nucleotides5-22 of SEQ ID NO.: 75, nucleotides 5-22 of SEQ ID NO.: 76, nucleotides5-29 of SEQ ID NO.: 77, nucleotides 6-26 of SEQ ID NO.: 78, nucleotides5-29 of SEQ ID NO.: 79, nucleotides 5-22 of SEQ ID NO.: 80, nucleotides5-22 of SEQ ID NO.: 81, nucleotides 4-22 of SEQ ID NO.: 82, nucleotides6-28 of SEQ ID NO.: 83, nucleotides 6-25 of SEQ ID NO.: 84, nucleotides3-23 of SEQ ID NO.: 85, and nucleotides 2-24 of SEQ ID NO.: 95. Infurther implementations, the labeled polynucleotide can comprise asequence elected from the group consisting of SEQ ID NO.: 55, SEQ IDNO.: 56, SEQ ID NOS.: 75 through 85, and SEQ ID NO.: 95. In certainimplementations, the sequence of the labeled polynucleotide is selectedfrom the group consisting of SEQ ID NO.: 55, SEQ ID NO.: 56, SEQ IDNOS.: 75 through 85, and SEQ ID NO.: 95.

In certain implementations, the composition targets the first openreading frame of SARS-CoV2 (ORF1ab). In such implementation, the set ofpolynucleotides can be Set-11 or Set-13, and the labeled polynucleotidecomprises a sequence selected from the group consisting of nucleotides5-29 of SEQ ID NO.: 77 and nucleotides 5-29 of SEQ ID NO.: 79. In someembodiments, the labeled polynucleotide comprises SEQ ID NO.: 79, ormore preferably SEQ ID NO.: 77. Preferably, the set of polynucleotidesis Set-11. In another implementation targeting ORF1ab, the set ofpolynucleotides is Set-12 and the labeled polynucleotide comprisesnucleotides 6-26 of SEQ ID NO.: 78. In some implementations, the labeledpolynucleotide comprises SEQ ID NO.: 78.

In certain implementations, the composition targets gene N, whichencodes a nucleoprotein that packages the positive strand viral genomeRNA into a helical ribonucleocapsid (RNP) and plays a fundamental roleduring virion assembly through its interactions with the viral genomeand membrane protein M. In such implementations, the set ofpolynucleotides can be Set-5 or Set-9 and the labeled polynucleotidecomprises a sequence selected from the group consisting of nucleotides5-22 of SEQ ID NO.: 75, nucleotides 5-22 of SEQ ID NO.: 76, nucleotides5-22 of SEQ ID NO.: 80, nucleotides 5-22 of SEQ ID NO.: 81, nucleotides4-22 of SEQ ID NO.: 82, nucleotides 6-28 of SEQ ID NO.: 83, nucleotides6-25 of SEQ ID NO.: 84, nucleotides 3-23 of SEQ ID NO.: 85. In someembodiments, the labeled polynucleotide comprises a sequence selectedfrom the group consisting of SEQ ID NO.: 75, SEQ ID NO.: 76, SEQ ID NO.:80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ ID NO.:83, SEQ ID NO.: 84, andSEQ ID NO.: 85. Preferably, the set of polynucleotides is Set-9.Similarly, the sequence of the labeled polynucleotides preferably is SEQID NO.: 83. In a second embodiment targeting the N gene, the set ofpolynucleotides is Set-1 or Set-4 and the labeled polynucleotidecomprises nucleotides 5-27 of SEQ ID NO.: 55. In such embodiments, thelabeled polynucleotide preferably comprises SEQ ID NO.: 55. In yetanother embodiment targeting the N gene, the set of polynucleotides isSet-3 or Set-8 and the labeled polynucleotide comprises nucleotides 7-25of SEQ ID NO.: 56. More preferably, the labeled polynucleotide comprisesSEQ ID NO.: 56.

In certain implementations, the composition targets gene E, whichencodes an envelope protein. In such implementations, the set ofpolynucleotides is selected from the group consisting of Set-14, Set-15,Set-16, and Set-17, and the labeled polynucleotide comprises nucleotides2-24 of SEQ ID NO.: 95. More preferably, the labeled polynucleotidecomprises SEQ ID NO.: 95. In a preferred implementation, thepolynucleotide sequence of the molecular beacon consists of SEQ IDNO.:95.

In certain implementations, the composition for detecting the presenceof SARS-CoV-2 comprises SEQ ID NO.: 49, SEQ ID NO.: 50, SEQ ID NO.: 51,SEQ ID NO.: 52, SEQ ID NO.: 53, and SEQ ID NO.: 54. In such animplementation, the composition can further comprise a molecular beaconcomprising a fluorophore, a quencher, and SEQ ID NO. 83. In anotherimplementation, the composition further comprises SEQ ID NO.: 63, SEQ IDNO.: 64, SEQ ID NO.: 65, SEQ ID NO.: 66, SEQ ID NO.: 67, and SEQ ID NO.:68.

In yet another embodiment, the composition for detecting the presence ofSARS-COV-2 comprises SEQ ID NO.: 63, SEQ ID NO.: 64, SEQ ID NO.: 65, SEQID NO.: 66, SEQ ID NO.: 67, and SEQ ID NO.: 68. This composition canfurther comprise a molecular beacon comprising a fluorophore, a quencherand SEQ ID NO. 77.

Yet another aspect of the invention provides a composition comprising afirst set of polynucleotides comprising SEQ ID NO.: 49, SEQ ID NO.: 50,SEQ ID NO.: 51, SEQ ID NO.: 52, SEQ ID NO.: 53 and SEQ ID NO.: 66; and asecond set of polynucleotides comprising SEQ ID NO.: 63, SEQ ID NO.: 64,SEQ ID NO.: 65, SEQ ID NO.: 66, SEQ ID NO.: 67 and SEQ ID NO.: 68. Insome implementations, the composition further comprises a first labeledpolynucleotide comprising nucleotides 6-28 of SEQ ID NO.: 83 and asecond labeled polynucleotide comprising nucleotides 5-29 of SEQ ID NO.:77. In some implementations, the first polynucleotide comprises SEQ IDNO.: 83 and the second labeled polynucleotide comprising SEQ ID NO.: 77.

In many implementations described herein, the probe is a molecularbeacon comprising a fluorophore, a quencher, and a polynucleotide. Inimplementations wherein the set of polynucleotides is Set-5 or Set-9,the molecular beacon comprises a sequence selected from the groupconsisting of nucleotides 5-22 of SEQ ID NO.: 75, nucleotides 5-22 ofSEQ ID NO.: 76, nucleotides 5-22 of SEQ ID NO.: 80, nucleotides 5-22 ofSEQ ID NO.: 81, nucleotides 4-22 of SEQ ID NO.: 82, nucleotides 6-28 ofSEQ ID NO.: 83, nucleotides 6-25 of SEQ ID NO.: 84, and nucleotides 3-23of SEQ ID NO.: 85. More preferably, the molecular beacon comprises asequence selected from the group consisting of SEQ ID NO.: 75, SEQ IDNO: 76, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ ID NO.: 83,SEQ ID NO.: 84 and SEQ ID NO.: 85. Even more preferably, thepolynucleotide sequence of the molecular beacon consists of a sequenceselected from the group consisting of SEQ ID NO.: 75, SEQ ID NO: 76, SEQID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ ID NO.: 83, SEQ ID NO.:84 and SEQ ID NO.: 85. In other implementations, wherein the set ofpolynucleotides is Set-1 or Set-4, the molecular beacon comprisesnucleotides 5-27 of SEQ ID NO.: 55, and more preferably comprises SEQ IDNO.: 55. In a preferred implementation, the polynucleotide sequence ofthe molecular beacon consists of SEQ ID NO.: 55. In implementationswherein the set of polynucleotides is Set-3 or Set-8, the molecularbeacon comprises nucleotides 7-25 of SEQ ID NO.: 56, more preferably thefull sequence SEQ ID NO.: 56. In a preferred implementation, thepolynucleotide sequence of the molecular beacon consists of SEQ ID NO.:56. In implementations, wherein the set of polynucleotides is Set-11 orSet-13, the molecular beacon comprises a sequence selected from thegroup consisting of nucleotides 5-29 of SEQ ID NO.: 77 and nucleotides5-29 of SEQ ID NO.: 79. Preferably, the molecular beacon comprises asequence selected from the group consisting of SEQ ID NO.: 77 and SEQ IDNO.: 79. In a particularly preferred implementation, the polynucleotidesequence of the molecular beacon consists of SEQ ID NO.: 77 or SEQ IDNO.: 79. In implementations wherein the set of polynucleotides isSet-12, the molecular beacon comprises nucleotides 6-26 of SEQ ID NO.:78, more preferably the full sequence of SEQ ID NO.: 78. In a preferredimplementation, the polynucleotide sequence of the molecular beaconconsists of SEQ ID NO.: 78. In implementations, wherein the set ofpolynucleotides is Set-14, Set-15, Set16 or Set-17, the molecular beaconcomprises nucleotides 2-24 of SEQ ID NO.: 95, more preferably the fullsequence of SEQ ID NO.: 95. In a preferred implementation, Thepolynucleotide sequence of the molecule beacon consists of SEQ ID NO.:95.

One aspect of the invention provides molecular beacons comprising afluorophore, a quencher, and a polynucleotide, wherein thepolynucleotide comprises a sequence selected from the group consistingof nucleotides 5-27 of SEQ ID NO.: 55, nucleotides 7-25 of SEQ ID NO.:56, nucleotides 5-22 of SEQ ID NO.: 75, nucleotides 5-22 of SEQ ID NO.:76, nucleotides 5-29 of SEQ ID NO.: 77, nucleotides 6-26 of SEQ ID NO.:78, nucleotides 5-29 of SEQ ID NO.: 79, nucleotides 5-22 of SEQ ID NO.:80, nucleotides 5-22 of SEQ ID NO.: 81, nucleotides 4-22 of SEQ ID NO.:82, nucleotides 6-28 of SEQ ID NO.: 83, nucleotides 6-25 of SEQ ID NO.:84, nucleotides 3-23 of SEQ ID NO.: 85, and nucleotides 2-24 of SEQ IDNO.: 95. In a preferred implementation, the polynucleotide portion ofthe molecular beacon comprises a sequence selected from the groupconsisting of SEQ ID NO.: 55, SEQ ID NO.: 56, SEQ ID NOS.: 75 through85, and SEQ ID NO.: 95. More preferably, the sequence selected from thegroup consisting of SEQ ID NO.: 55, SEQ ID NO.: 56, SEQ ID NOS.: 75through 85, and SEQ ID NO.: 95.

Yet another aspect of the invention provides method of detectingSARS-CoV-2 in a test sample, the method comprising (a) extractingnucleic acid from the test sample, (b) amplifying a target sequence byreacting the nucleic acid extracted in step (a) with a reaction mixturecomprising a strand displacement DNA polymerase and a reversetranscriptase and a sequence specific primer set, wherein saidsequence-specific primer set is selected from the group consisting ofSet-1 through Set-17, and (c) detecting the presence or absence of anamplified product of step (b); wherein the presence of saidamplification product is indicative of the presence of SARS-CoV-2 in thetest sample. In one embodiment, the amplification in step (b) of thetarget sequence is performed between about 60° C. and about 67° C. forless than 30 minutes. Preferably, the amplification step is performedfor less than fifteen minutes. In some implementations, the stranddisplacement DNA polymerase and the reverse transcriptase activities areprovided by a single enzyme.

In certain embodiments, detecting the presence or absence of theamplification product comprises hybridizing the amplified product with aprobe comprising a polynucleotide attached to a label. In a preferredimplementation, the label is a fluorophore, which is preferably attachedto a terminus of the polynucleotide. In a particularly preferredembodiment, the probe or polynucleotide is a molecular beacon comprisinga fluorophore, a quencher, and a polynucleotide. In one embodiment, thefluorophore is FAM and the quencher is BHQ1. In an alternateimplementation, the fluorophore is ATTO 565 or Alexa 594 or Cy5 and thequencher is BHQ1 or BHQ2. In other implementations, detecting thepresence or absence of the amplification product comprises exposing theamplified product to an intercalating dye.

Another aspect of the invention provides methods of detecting SARS-CoV-2in a test sample, the method comprising (a) extracting nucleic acid fromthe test sample, (b) amplifying a target sequence by reacting nucleicacid extracted in step (a) for less than ten minutes with a reactionmixture comprising a strand displacement DNA polymerase and a sequencespecific LAMP primer set, and (c) detecting the presence or absence ofan amplified product of step (b); wherein the presence of saidamplification product is indicative of the presence of SARS-CoV-2 in thetest sample. In some implementations, the amplifying step comprisesreacting the nucleic acid extracted in step (a) with a reaction mixturecomprising a strand displacement DNA polymerase and a sequence-specificprimer set, wherein said sequence-specific primer set is selected fromthe group consisting of Set-1 through Set-17. In such implementations,detecting the presence or absence of the amplification product cancomprise hybridizing the amplified product with a molecular beaconcomprising a polynucleotide sequence selected from the group consistingof nucleotides 5-27 of SEQ ID NO.: 55, nucleotides 7-25 of SEQ ID NO.:56, nucleotides 5-22 of SEQ ID NO.: 75, nucleotides 5-22 of SEQ ID NO.:76, nucleotides 5-29 of SEQ ID NO.: 77, nucleotides 6-26 of SEQ ID NO.:78, nucleotides 5-29 of SEQ ID NO.: 79, nucleotides 5-22 of SEQ ID NO.:80, nucleotides 5-22 of SEQ ID NO.: 81, nucleotides 4-22 of SEQ ID NO.:82, nucleotides 6-28 of SEQ ID NO.: 83, nucleotides 6-25 of SEQ ID NO.:84, nucleotides 3-23 of SEQ ID NO.: 85, and nucleotides 2-24 of SEQ IDNO.: 95. In such implementations, detecting the presence or absence ofthe amplification product can comprise hybridizing the amplified productwith a molecular beacon consisting of a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO.: 55, SEQ ID NO.: 56, SEQ IDNOS.: 75 through 85, and SEQ ID NO.: 95.

In certain implementations of the method to detect SARS-CoV-2 in a testsample, the set of polynucleotides is Set-1 or Set-4, and the labeledpolynucleotide comprises nucleotides 5-27 of SEQ ID NO.: 55. Morepreferably, the sequence of the labeled polynucleotides is SEQ ID NO.:55. In implementations wherein the set of polynucleotides is Set-3 orSet-8, the labeled polynucleotide comprises nucleotides 7-25 of SEQ IDNO.: 56. More preferably, the sequence of the labeled polynucleotides isSEQ ID NO.: 56. In implementations, wherein the set of polynucleotidesis Set-5 or Set-9, the labeled polynucleotide comprises a sequenceselected from the group consisting of nucleotides 5-22 of SEQ ID NO.:75, nucleotides 5-22 of SEQ ID NO.: 76, nucleotides 5-22 of SEQ ID NO.:80, nucleotides 5-22 of SEQ ID NO.: 81, nucleotides 4-22 of SEQ ID NO.:82, nucleotides 6-28 of SEQ ID NO.: 83, nucleotides 6-25 of SEQ ID NO.:84, nucleotides 3-23 of SEQ ID NO.: 85. More preferably, the sequence ofthe labeled polynucleotide is selected from the group consisting of SEQID NO.: 75, SEQ ID NO.: 76, SEQ ID NO.: 80, SEQ ID NO.: 82, SEQ ID NO.:83, SEQ ID NO.: 84, and SEQ ID NO.: 85. In implementations wherein theset of polynucleotides is Set-9, the sequence of the labeledpolynucleotide preferably is SEQ ID NO.: 83. In implementations whereinthe set of polynucleotides is Set-11 or Set-13, the labeledpolynucleotide comprises a sequence selected from the group consistingof nucleotides 5-29 of SEQ ID NO.: 77 and nucleotides 5-29 of SEQ IDNO.: 79. Preferably, the labeled polynucleotide comprises SEQ ID NO.: 77or SEQ ID NO.: 79. In implementations, wherein the set ofpolynucleotides is Set-12, the labeled polynucleotide comprisesnucleotides 6-26 of SEQ ID NO.: 78. Preferably, the sequence of thelabeled polynucleotides is SEQ ID NO.: 78. In implementations whereinthe set of polynucleotides is selected from the group consisting ofSet-14, Set-15, Set-16 and Set-17, the labeled polynucleotide comprisesnucleotides 2-24 of SEQ ID NO.: 95. More preferably, the sequence of thelabeled polynucleotides is SEQ ID NO.: 95.

Yet another aspect of the invention provides kits comprising thecompositions comprising a set of polynucleotides selected from the groupconsisting Set-1 through Set-17. In some embodiments, the kit furthercomprises a strand displacement polymerase and a reverse transcriptase.In certain embodiments, the kit comprises a molecular beacon comprisinga fluorophore, a quencher, and a polynucleotide, wherein thepolynucleotide comprises a sequence selected from the group consistingof nucleotides 5-27 of SEQ ID NO.: 55, nucleotides 7-25 of SEQ ID NO.:56, nucleotides 5-22 of SEQ ID NO.: 75, nucleotides 5-22 of SEQ ID NO.:76, nucleotides 5-29 of SEQ ID NO.: 77, nucleotides 6-26 of SEQ ID NO.:78, nucleotides 5-29 of SEQ ID NO.: 79, nucleotides 5-22 of SEQ ID NO.:80, nucleotides 5-22 of SEQ ID NO.: 81, nucleotides 4-22 of SEQ ID NO.:82, nucleotides 6-28 of SEQ ID NO.: 83, nucleotides 6-25 of SEQ ID NO.:84, nucleotides 3-23 of SEQ ID NO.: 85, and nucleotides 2-24 of SEQ IDNO.: 95. The polynucleotide sequence of the molecular beacon cancomprise a sequence selected from the group consisting of SEQ ID NO.:55, SEQ ID NO.: 56, SEQ ID NOS.: 75 through 85, and SEQ ID NO.: 95. Insome embodiments, the polynucleotide sequence of the molecular beaconconsists of a sequence selected from the group consisting of SEQ ID NO.:55, SEQ ID NO.: 56, SEQ ID NOS.: 75 through 85, and SEQ ID NO.: 95.

In certain implementations of the kit, the set of polynucleotides isSet-1 or Set-4. In such implementation, the kit can further comprise amolecular beacon comprising a fluorophore, a quencher, and apolynucleotide, wherein the polynucleotide comprises nucleotides 5-27 ofSEQ ID NO.: 55. Preferably, the polynucleotide of the molecular beaconconsists of SEQ ID NO.: 55. In other implementations of the kit, the setof polynucleotides is selected from the group consisting of Set-3 andSet-8. In such implementations, the kit can further comprise a molecularbeacon comprising a fluorophore, a quencher, and a polynucleotide,wherein the polynucleotide comprises nucleotides 7-25 of SEQ ID NO.: 56.Preferably, the polynucleotide of the molecular beacon consists of SEQID NO.: 56. In yet another implementation of the kit, the set ofpolynucleotides is selected from the group consisting of Set-5 andSet-9. In such implementations, the kit can further comprise a molecularbeacon comprising a fluorophore, a quencher, and a polynucleotide,wherein the polynucleotide comprises a sequence selected from the groupconsisting of nucleotides 5-22 of SEQ ID NO.: 75, nucleotides 5-22 ofSEQ ID NO.: 76, nucleotides 5-22 of SEQ ID NO.: 80, nucleotides 5-22 ofSEQ ID NO.: 81, nucleotides 4-22 of SEQ ID NO.: 82, nucleotides 6-28 ofSEQ ID NO.: 83, nucleotides 6-25 of SEQ ID NO.: 84, and nucleotides 3-23of SEQ ID NO.: 85. Preferably, the polynucleotide of the molecularbeacon consists of a sequence selected from the group consisting of SEQID NO.: 75, SEQ ID NO.: 76, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.:82, SEQ ID NO.: 83, SEQ ID NO.: 84, and SEQ ID NO.: 85. In otherimplementations of the kit, the set of polynucleotides is selected fromthe group consisting of Set-11 and Set-13. In such implementations, thekit can further comprise a molecular beacon comprising a fluorophore, aquencher, and a polynucleotide, wherein the polynucleotide comprises asequence selected from the group consisting of nucleotides 5-29 of SEQID NO.: 77 and nucleotides 5-29 of SEQ ID NO.: 79. Preferably, thepolynucleotide of the molecular beacon consists of a sequence selectedfrom the group consisting of SEQ ID NO.: 77 and SEQ ID NO.: 79. In yetanother implementation of the kit, the set of polynucleotides is Set-12.In such implementations, the kit can further comprise a molecular beaconcomprising a fluorophore, a quencher, and a polynucleotide, wherein thepolynucleotide comprises nucleotides 6-26 of SEQ ID NO.: 78. Preferably,the polynucleotide of the molecular beacon consists of SEQ ID NO.: 78.In certain implementations of the kit, the set of polynucleotides isselected from the group consisting of Set-14, Set-15, Set-16, andSet-17. In such implementations, the kit can further comprise amolecular beacon comprising a fluorophore, a quencher, and apolynucleotide comprising nucleotides 2-24 of SEQ ID NO.: 95.Preferably, the polynucleotide of the molecular beacon consists of SEQID NO.: 95.

Another aspect of the invention provides a kit comprising a set ofpolynucleotides comprising SEQ ID NO.: 49, SEQ ID NO.: 50, SEQ ID NO.:51, SEQ ID NO.: 52, SEQ ID NO.: 53, and SEQ ID NO.: 54. The kitpreferably, further comprises a strand displacement polymerase andreverse transcriptase. In some implementations, the kit furthercomprises a molecular beacon comprising a fluorophore, a quencher, andSEQ ID NO. 83. In another implementation, the kit further comprises aset of polynucleotides comprising SEQ ID NO.: 63, SEQ ID NO.: 64, SEQ IDNO.: 65, SEQ ID NO.: 66, SEQ ID NO.: 67, and SEQ ID NO.: 68. This kitpreferably further comprises a molecular beacon comprising afluorophore, a quencher and SEQ ID NO. 77.

DETAILED DESCRIPTION

The present invention encompasses, in some embodiments, a compositioncomprising a set of polynucleotides for priming a nucleic acidamplification reaction and methods of using such. In some embodiments,the composition further comprises a probe.

As used herein, “nucleic acid” includes both DNA and RNA, including DNAand RNA containing non-standard nucleotides. A “nucleic acid” containsat least one polynucleotide (a “nucleic acid strand”). A “nucleic acid”may be single-stranded or double-stranded. The term “nucleic acid”refers to nucleotides and nucleosides which make up, for example,deoxyribonucleic acid (DNA) macromolecules and ribonucleic acid (RNA)macromolecules. The most common nucleic acids are deoxyribonucleic acid(DNA) and ribonucleic acid (RNA). It should be further understood thatthe present invention can be used for biological sequences containingartificial nucleotides such as peptide nucleic acid (PNA), morpholino,locked nucleic acid (LNA), glycol nucleic acid (GNA) and threose nucleicacid (TNA), among others. Preferably, the artificial nucleotides arelocked nucleic acid molecules, including [alpha]-L-LNAs. LNAs compriseribonucleic acid analogues wherein the ribose ring is “locked” by amethylene bridge between the 2′-oxygen and the 4′-carbon—i.e.,oligonucleotides, containing at least one LNA monomer, that is, one2′-O,4′-C-methylene-β-D-ribofuranosyl nucleotide. LNA bases formstandard Watson-Crick base pairs but the locked configuration increasesthe rate and stability of the basepairing reaction (Jepsen et al.,Oligonucleotides, 14, 130-146 (2004)).

As used herein, a “polynucleotide” refers to a polymeric chaincontaining two or more nucleotides, which contain deoxyribonucleotides,ribonucleotides, and/or their analog, such as those containing modifiedbackbones (e.g. peptide nucleic acids (PNAs) or phosphorothioates) ormodified bases. “Polynucleotides” includes primers, oligonucleotides,nucleic acid strands, etc. A polynucleotide may contain standard ornon-standard nucleotides. Thus, the term includes mRNA, tRNA, rRNA,ribozymes, DNA, cDNA, recombinant nucleic acids, branched nucleic acids,plasmids, vectors, probes, primers, etc. Typically, a polynucleotidecontains a 5′ phosphate at one terminus (“5′ terminus”) and a 3′hydroxyl group at the other terminus (“3′ terminus”) of the chain. Themost 5′ nucleotide of a polynucleotide may be referred to herein as the“5′ terminal nucleotide” of the polynucleotide. The most 3′ nucleotideof a polynucleotide may be referred to herein as the “3′ terminalnucleotide” of the polynucleotide. Where nucleic acid of the inventiontakes the form of RNA, it may or may not have a 5′ cap.

LAMP is a nucleic acid amplification method that relies on auto-cyclestrand-displacement DNA synthesis performed by Bst DNA polymerase, orother strand displacement polymerases. The amplified products arestem-loop structures with several repeated sequences of the target andhave multiple loops. The principal merit of this method is thatdenaturation of the DNA template is not required, and thus the LAMPreaction can be conducted under isothermal conditions (ranging from 60to 67° C.). LAMP requires only one enzyme and four types of primers thatrecognize six distinct hybridization sites in the target sequence. Thereaction can be accelerated by the addition of two additional primers.The method produces a large amount of amplified product, resulting ineasier detection, such as detection by visual judgment of the turbidityor fluorescence of the reaction mixture.

In brief, the reaction is initiated by annealing and extension of a pairof ‘loop-forming’ primers (forward and backward inner primers, FIP andBIP, respectively), followed by annealing and extension of a pair offlanking primers (F3 and B3). Extension of these primers results instrand-displacement of the loop-forming elements, which fold up to formterminal hairpin-loop structures. Once these key structures haveappeared, the amplification process becomes self-sustaining, andproceeds at constant temperature in a continuous and exponential manner(rather than a cyclic manner, like PCR) until all of the nucleotides(dATP, dTTP, dCTP & dGTP) in the reaction mixture have been incorporatedinto the amplified DNA. Optionally, an additional pair of primers can beincluded to accelerate the reaction. These primers, termed Loop primers,hybridize to non-inner primer bound terminal loops of the inner primerdumbbell shaped products.

The term “primer” as used herein refers to an oligonucleotide, which iscapable of acting as a point of initiation of synthesis when placedunder conditions in which synthesis of primer extension product which iscomplementary to a nucleic acid strand (template) is induced, i.e., inthe presence of nucleotides and an agent for polymerization, such as DNApolymerase, and at a suitable temperature and pH.

Applications for LAMP have been further extended to include detection ofRNA molecules by addition of Reverse Transcriptase enzyme (RT). Byincluding RNA detection, the types of targets for which LAMP can beapplied are also expanded and add the ability to additionally target RNAbased viruses, important regulatory non-coding RNA (sRNA, miRNA), andRNA molecules that have been associated with particular disease orphysiological states. The ability to detect RNA also has the potentialto increase assay sensitivity, for instance in choosing highlyexpressed, stable, and/or abundant messenger RNA (mRNA) or ribosomal RNA(rRNA) targets. This preliminary phase of amplification involves thereverse transcription of RNA molecules to complementary DNA (cDNA). ThecDNA then serves as template for the strand displacing DNA polymerase.Use of a thermostable RT enzyme (i.e., NEB RTx) enables the reaction tobe completed at a single temperature and in a one step, single mixreaction.

A “target sequence,” as used herein, means a nucleic acid sequence ofNeisseria gonorrhoeae, or complement thereof, that is amplified,detected, or both amplified and detected using one or more of thepolynucleotides herein provided. Additionally, while the term targetsequence sometimes refers to a double stranded nucleic acid sequence,those skilled in the art will recognize that the target sequence canalso be single stranded, e.g., RNA. A target sequence may be selectedthat is more or less specific for a particular organism. For example,the target sequence may be specific to an entire genus, to more than onegenus, to a species or subspecies, serogroup, auxotype, serotype,strain, isolate or other subset of organisms.

The speed, specificity and sensitivity of the primers/probe compositionsand method described herein result from several aspects. Exemplaryprimers for use in the compositions and methods according to the presentinvention include those provided in Table 1.

TABLE 1 Primer Sequences Sequence ID Target Sequence (5′ to 3′)SEQ ID No.: 1 N gene GGACCAGGAACTAATCAGACA SEQ ID No.: 2 N geneTCTGCGGTAAGGCTTGAG SEQ ID No.: 3 N geneACCACGTTCCCGAAGGTGTCAGCGTTCTTCGGAATGTC SEQ ID No.: 4 N geneTGACCTACACAGGTGCCATCAAGGCTCTGTTGGTGGGAAT SEQ ID No.: 5 N geneGACTTCCATGCCAATGCG SEQ ID No.: 6 N gene GCTGAATAAGCATATTGACGCATACSEQ ID No.: 7 N gene ACCCCAAAATCAGCGAAA SEQ ID No.: 8 N geneATTGGAACGCCTTGTCC SEQ ID No.: 9 N gene ATCGCGCCCCACTGCCACCCCGCATTACGTTTSEQ ID No.: 10 N gene CGTCGGCCCCAAGGTTTATCCTTGCCATGTTGAGTGSEQ ID No.: 11 N gene CAGTTGAATCTGAGGGTCCA SEQ ID No.: 12 N geneGTCTTGGTTCACCGCTCT SEQ ID No.: 13 N gene AAAAGGCTTCTACGCAGAASEQ ID No.: 14 N gene CCTTTACCAGACATTTTGCTC SEQ ID No.: 15 N geneACTGCTGCCTGGAGTTGAATCAGTCAAGCCTCTTCTCG SEQ ID No.: 16 N geneCTGCTAGAATGGCTGGCAATGTGGTTCAATCTGTCAAGCA SEQ ID No.: 17 N geneACTGTTGCGACTACGTGAT SEQ ID No.: 18 N gene GCGGTGATGCTGCTCTSEQ ID No.: 19 N gene AGGAACTGATTACAAACATTGG SEQ ID No.: 20 N geneTTTTGTATGCGTCAATATGCTT SEQ ID No.: 21 N geneAAGGTGTGACTTCCATGCCAACAATTTGCCCCCAGC SEQ ID No.: 22 N geneGGGAACGTGGTTGACCTACAGACTTGATCTTTGAAATTTGGATC SEQ ID No.: 23 N geneTGCGCGACATTCCGAA SEQ ID No.: 24 N gene GGTGCCATCAAATTGGATGASEQ ID No.: 25 N gene TCAAAGATCAAGTCATTTTGCT SEQ ID No.: 26 N geneGCCTGAGTTGAGTCAGC SEQ ID No.: 27 N geneGTCTCTGCGGTAAGGCTTGAATACAAAACATTCCCACCAAC SEQ ID No.: 28 N geneGCAAACTGTGACTCTTCTTCCTGTGCTCATGGATTGTTGCA SEQ ID No.: 29 N geneATCAGCCTTCTTCTTTTTGTCC SEQ ID No.: 30 N gene TGCAGATTTGGATGATTTCTCCSEQ ID No.: 31 ORF1ab CAGAAATCAATACTGAGTCCTCT SEQ ID No.: 32 ORF1abGTAGCCAAATCAGATGTGAAC SEQ ID No.: 33 ORF1abCACAGAATTTTGAGCAGTTTCAAGAGTTCAGAGGCTGCTCGTG SEQ ID No.: 34 ORF1abCGTGTTTTACAGAAGGCCGCCATAGCATCAATGAGTCTCAGT SEQ ID No.: 35 ORF1abGCGGGAGAAAATTGATCGTAC SEQ ID No.: 36 ORF1ab ATAACAATACTAGATGGAATTTCACAGTSEQ ID No.: 37 ORF1ab CAAATTGTTGAATCCTGTGGT SEQ ID No.: 38 ORF1abAAATTCCATCTAGTATTGTTATAGCG SEQ ID No.: 39 ORF1abAATGCATAAAGAGGACTCAGTATTGATTTCAATTTTAAAGTTACAAAAGGAAAAGCT SEQ ID No.: 40ORF1ab CAGAGGCTGCTCGTGTTGTCACGCACAGAATTTTGAGC SEQ ID No.: 41 ORF1abTGTTCACCAATATTCCAGGCA SEQ ID No.: 42 ORF1ab CGATCAATTTTCTCCCGCACSEQ ID No.: 43 N gene GTCAAGCCTCTTCTCGTTC SEQ ID No.: 44 N geneCTTAGTGACAGTTTGGCCTT SEQ ID No.: 45 N geneCCGCCATTGCCAGCCAAACAGTTCAAGAAATTCAACTCC SEQ ID No.: 46 N geneGATGCTGCTCTTGCTTTGCTGGCCTTTACCAGACATTTTGC SEQ ID No.: 47 N geneGAGAAGTTCCCCTACTGCTG SEQ ID No.: 48 N gene TGACAGATTGAACCAGCTTGASEQ ID No.: 49 N gene AATTTCAAAGATCAAGTCATTTTGC SEQ ID No.: 50 N geneGTTGAGTCAGCACTGCTC SEQ ID No.: 51 N geneAAGGCTTGAGTTTCATCAGCCTCGCATACAAAACATTCCCAC SEQ ID No.: 52 N geneGCAGAGACAGAAGAAACAGCAAACATTGTTGCAATTGTTTGGAGAA SEQ ID No.: 53 N geneTCTTTTTGTCCTTTTTAGGCTCTG SEQ ID No.: 54 N gene CTCTTCTTCCTGCTGCAGATTTGGSEQ ID No.: 57 ORF1ab CAAATTGTTGAATCCTGTGGT SEQ ID No.: 58 ORF1abAAATTCCATCTAGTATTGTTATAGCG SEQ ID No.: 59 ORF1abAATGCATAAAGAGGACTCAGTATTGATTTCAATTTTAAAGTTACAAAAGGAAAAGCT SEQ ID No.: 60ORF1ab CAGAGGCTGCTCGTGTTGTCACGCACAGAATTTTGAGC SEQ ID No.: 61 ORF1abTGTTCACCAATATTCCAGGCA SEQ ID No.: 62 ORF1ab CGATCAATTTTCTCCCGCACSEQ ID No.: 63 ORF1ab TCTTATCAGAGGCACGTCA SEQ ID No.: 64 ORF1abTGTCTCACCACTACGACC SEQ ID No.: 65 ORF1abACGTTTGATGAACACATAGGGCTTAAAGATGGCACTTGTGGC SEQ ID No.: 66 ORF1abTCGGATGCTCGAACTGCACTGCCTTCGAGTTCTGCTA SEQ ID No.: 67 ORF1abTTCAAGTTGAGGCAAAACGC SEQ ID No.: 68 ORF1ab CATGGTCATGTTATGGTTGAGCSEQ ID No.: 69 ORF1ab ATGTCCAAATTTTGTATTTCCCTT SEQ ID No.: 70 ORF1abGCTTTAACAAAATCGCCCG SEQ ID No.: 71 ORF1abGCAACTGGATAGACAGATCGAATTCTATCCATAATCAAGACTATTCAACCA SEQ ID No.: 72ORF1ab CACCAAATGAATGCAACCAAATGTGTCTGCCATGAAGTTTCACC SEQ ID No.: 73ORF1ab CATCAAGCTTTTTCTTTTCAACCC SEQ ID No.: 74 ORF1abCCTTTCAACTCTCATGAAGTGTG SEQ ID No.: 86 ORF1ab GAGGGACAAGGACACCAAGSEQ ID No.: 87 ORF1ab TCGGATGCTCGAACTGCACTGTCTCACCACTACGACCSEQ ID No.: 88 ORF1ab GGTAGCAGAACTCGAAGGCAT SEQ ID No.: 89 E geneAAGAGACAGGTACGTTAATAGT SEQ ID No.: 90 E gene TTAGACCAGAAGATCAGGAACSEQ ID No.: 91 E gene AAGCGCAGTAAGGATGGCTATAGCGTACTTCTTTTTCTTGCSEQ ID No.: 92 E gene TGCGTACTGCTGCAATATTGTTTTAACACGAGAGTAAACGTAAASEQ ID No.: 93 E gene TGTAACTAGCAAGAATACCACG SEQ ID No.: 94 E geneCGTGAGTCTTGTAAAACCTTCT SEQ ID No.: 96 E gene GAAGATCAGGAACTCTAGAAGASEQ ID No.: 97 E gene ACACAATCGAAGCGCAGTAAGTTTTCTTGCTTTCGTGGTATSEQ ID No.: 98 E gene GCGTACTGCTGCAATATTGTTTTAACACGAGAGTAAACGTAAASEQ ID No.: 99 E gene TGGCTAGTGTAACTAGCAAGA SEQ ID No.: 100 E geneACGTTAATAGTTAATAGCGTACTT SEQ ID No.: 101 E gene TCAGGAACTCTAGAAGAATTCASEQ ID No.: 102 E gene ACAATCGAAGCGCAGTAAGGTTTTCTTGCTTTCGTGGTATSEQ ID No.: 103 E gene TGCGTACTGCTGCAATATTGTTTTTAACACGAGAGTAAACGTAAA

Detection of the LAMP amplified products can be achieved via a varietyof methods. In some implementations, LAMP amplified products aredetected using intercalating dyes. Intercalating dyes are generallyaromatic cations with planar structures that insert between stacked basepairs in the DNA duplex, an arrangement that provides an environmentallydependent fluorescence enhancement for dye molecules and creates a largeincrease in the fluorescence signal relative to the free dye insolution. The signal enhancement provides a proportional response,allowing direct quantitative DNA measurements. Preferred intercalatingdyes in the present disclosure include fluorescent dyes. The dye can bea cyanine or a non-cyanine intercalating die. In some cases, theintercalating dye is a cyanine dye. In some cases, the cyanine dye canbe Thiazole Orange, SYBR® (e.g. Sybr Green I, Sybr Green II, Sybr Gold,SYBR DX), Oil Green, CyQuant GR, SYTOX Green, SYTO9, SYT010, SYTO17,SYBR14, Oxazile Yellow, Thiazone Orange, SYTO, TOTO, YOYO, BOBO, andPOPO. In some cases, the dye is a non-cyanine dye. In some cases, thenon cyanine dye is pentacene, anthracene, naphthalene, ferrocene, methylviologen, tri-morpholino ammonium, propidium (e.g., propidium iodide) oranother aromatic or heteroaromatic derivative.

In a preferred embodiment, detection of product is conducted by adding afluorescently-labeled probe to the primer mix. The term used herein“probe” refers to a single-stranded nucleic acid molecule comprising aportion or portions that are complementary, or substantiallycomplementary, to a target sequence. In certain implementations, thefluorescently-labeled probe is a molecular beacon.

As used herein, “molecular beacon” refers to a single strandedhairpin-shaped oligonucleotide probe designed to report the presence ofspecific nucleic acids in a solution. A molecular beacon consists offour components; a stem, hairpin loop, end labelled fluorophore andopposite end-labelled quencher (Tyagi et al., (1998) NatureBiotechnology 16:49-53). When the hairpin-like beacon is not bound to atarget, the fluorophore and quencher lie close together and fluorescenceis suppressed. In the presence of a complementary target nucleotidesequence, the stem of the beacon opens to hybridize to the target. Thisseparates the fluorophore and quencher, allowing the fluorophore tofluoresce. Alternatively, molecular beacons also include fluorophoresthat emit in the proximity of an end-labelled donor.“Wavelength-shifting Molecular Beacons” incorporate an additionalharvester fluorophore enabling the fluorophore to emit more strongly.Current reviews of molecular beacons include Wang et al., 2009, AngewChem Int Ed Engl, 48(5):856-870; Cissell et al., 2009, Anal Bioanal Chem393(1):125-35; Li et al., 2008, Biochem Biophys Res Comm 373(4):457-61;and Cady, 2009, Methods Mol Biol 554:367-79. Exemplary probes for use inthe compositions and methods according to the present invention includethose provided in Table 2. In certain implementations, the probes mayinclude one or more linked nucleic acids (LNA) as indicated by “[+X]”,where X indicates the identity of the nucleobase. Bold indicates theportion of the molecular beacon that hybridizes to sequences found inthe target coronavirus genome.

TABLE 2 Probe Sequences ID Fluor Quench Sequence (5′ to 3′) Sequence IDMB1 FAM BHQ1 CACGCCAAA[+T]TT[+C]AAA[+G]AT[+C]AAGTCATGGCGTGSEQ ID NO.: 55 MB2 FAM BHQ1 CAGCTGCTTGA[+C]AGA[+T]TGA[+A]CCAGCAGCTGSEQ ID NO.: 56 MB3 FAM BHQ1 CACGGTGACT[+C]TT[+C]TT[+C]CTGCTCACCGTGSEQ ID NO.: 75 MB4 FAM BHQ1 CGAGTCCTGC[+T]GC[+A]GA[+T]TTGGACTCGSEQ ID NO.: 76 MB5 FAM BHQ1 CAGCTCATGG[+T]CAT[+G]TTAT[+G]GTTGAGCTGSEQ ID NO.: 77 MB6 FAM BHQ1 CACACTTT[+C]AA[+C]TC[+T]CA[+T]GAAGTGTGSEQ ID NO.: 78 MB7 FAM BHQ1 CAGCTCATGGTCATGTTATGGTTGAGCTG SEQ ID NO.: 79MB8 FAM BHQ1 CGAGTCCTGC[+T]GC[+A]GA[+T]TTGGACTCG SEQ ID NO.: 80 MB9 FAMBHQ1 CATGTCCTGC[+T]GC[+A]GA[+T]TTGGACATG SEQ ID NO.: 81 MB10 FAM BHQ1CACTTCCTGC[+T]GC[+A]GA[+T]TTGGAAGTG SEQ ID NO.: 82 MB11 FAM BHQ1CACGCGTGACT[+C]TTCTT[+C]CTGC[+T]GCAGACGCGTG SEQ ID NO.: 83 MB12 FAM BHQ1CACGCTTC[+T]TC[+C]TGC[+T]GC[+A]GA[+T]TTGAAGCGTG SEQ ID NO.: 84 MB13 FAMBHQ1 CAGACTCTTCT[+T]CC[+T]GC[+T]GCAGAGTCTG SEQ ID NO.: 85 MB14 FAM BHQ1CACGTGAGT[+C]TT[+G]TAA[+A]ACC[+T]TCTCACGTG SEQ ID NO.: 95

In one implementation, the molecular beacon comprises a fluorophore, aquencher, and a polynucleotide, wherein the polynucleotide comprises asequence selected from the group consisting of nucleotides 5-27 of SEQID NO.: 55, nucleotides 7-25 of SEQ ID NO.: 56, nucleotides 5-22 of SEQID NO.: 75, nucleotides 5-22 of SEQ ID NO.: 76, nucleotides 5-29 of SEQID NO.: 77, nucleotides 6-26 of SEQ ID NO.: 78, nucleotides 5-29 of SEQID NO.: 79, nucleotides 5-22 of SEQ ID NO.: 80, nucleotides 5-22 of SEQID NO.: 81, nucleotides 4-22 of SEQ ID NO.: 82, nucleotides 6-28 of SEQID NO.: 83, nucleotides 6-25 of SEQ ID NO.: 84, nucleotides 3-23 of SEQID NO.: 85. In one embodiment, the polynucleotide comprises a sequenceselected from the group consisting of SEQ ID NO.: 55, SEQ ID NO.: 56 andSEQ ID NOS.: 75-85. In another embodiment, the polynucleotide consistsof a sequence selected from the group consisting of SEQ ID NO.: 55, SEQID NO.: 56 and SEQ ID NOS.: 75-85.

The molecular beacon is preferably used in a composition also comprisinga set of sequence-specific LAMP primers. In one implementation, themolecular beacon comprises a sequence selected from the group consistingof nucleotides 5-27 of SEQ ID NO.: 55 and nucleotides 7-25 of SEQ IDNO.: 56. In such an implementation, the molecular beacon can comprise asequence selected from the group consisting of SEQ ID NO.: 55, SEQ IDNO.: 56 and SEQ ID NOS.: 75-85. More preferably, polynucleotide sequenceof the molecular beacon consists of a sequence selected from the groupconsisting of SEQ ID NO.: 55, SEQ ID NO.: 56 and SEQ ID NOS.: 75-85.

The term “label” as used herein means a molecule or moiety having aproperty or characteristic which is capable of detection and,optionally, of quantitation. A label can be directly detectable, aswith, for example (and without limitation), radioisotopes, fluorophores,chemiluminophores, enzymes, colloidal particles, fluorescentmicroparticles and the like; or a label may be indirectly detectable, aswith, for example, specific binding members. It will be understood thatdirectly detectable labels may require additional components such as,for example, substrates, triggering reagents, quenching moieties, light,and the like to enable detection and/or quantitation of the label. Whenindirectly detectable labels are used, they are typically used incombination with a “conjugate”. A conjugate is typically a specificbinding member that has been attached or coupled to a directlydetectable label. Coupling chemistries for synthesizing a conjugate arewell known in the art and can include, for example, any chemical meansand/or physical means that does not destroy the specific bindingproperty of the specific binding member or the detectable property ofthe label. As used herein, “specific binding member” means a member of abinding pair, i.e., two different molecules where one of the moleculesthrough, for example, chemical or physical means specifically binds tothe other molecule. In addition to antigen and antibody specific bindingpairs, other specific binding pairs include, but are not intended to belimited to, avidin and biotin; haptens and antibodies specific forhaptens; complementary nucleotide sequences; enzyme cofactors orsubstrates and enzymes; and the like.

The molecular beacon can be composed of nucleic acid only such as DNA orRNA, or it can be composed of a peptide nucleic acid (PNA) conjugate.The fluorophore can be any fluorescent organic dye or a single quantumdot. The quenching moiety desirably quenches the luminescence of thefluorophore. Any suitable quenching moiety that quenches theluminescence of the fluorophore can be used. A fluorophore can be anyfluorescent marker/dye known in the art. Examples of suitablefluorescent markers include, but are not limited to, Fam, Hex, Tet, Joe,Rox, Tamra, Max, Edans, Cy dyes such as Cy5, Fluorescein, Coumarin,Eosine, Rhodamine, Bodipy, Alexa, Cascade Blue, Yakima Yellow, LuciferYellow, Texas Red, and the family of ATTO dyes. A quencher can be anyquencher known in the art. Examples of quenchers include, but are notlimited to, DABCYL (4-(dimethylaminoazo)benzene-4-carboxylic acid), darkquenchers such as ECLIPSE Dark Quencher, ElleQuencher, Tamra(tetramethylrhodamine), Black Hole quenchers and QSY dyes. The skilledperson would know which combinations of dye/quencher are suitable whendesigning a probe. In an exemplary embodiment, fluorescein (FAM) is usedin conjunction with Blackhole Quencher™ (BHQ™). Binding of the molecularbeacon to amplified product can then be directly, visually assessed.Alternatively, the fluorescence level can be measured by spectroscopy inorder to improve sensitivity.

A variety of commercial suppliers produce standard and custom molecularbeacons, including Abingdon Health (UK), Attostar (US, MN), Biolegio(NLD), Biomers.net (DEU), Biosearch Technologies (US, CA), Eurogentec(BEL), Gene Link (US, NY) Integrated DNA Technologies (US, IA), IsogenLife Science (NLD), Midland Certified Reagent (US, TX), Eurofins (DEU),Sigma-Aldrich (US, TX), Thermo Scientific (US, MA), TIB MOLBIOL (DEU),TriLink Bio Technologies (US, CA). A variety of kits, which utilizemolecular beacons are also commercially available, such as the Sentinel™Molecular Beacon Allelic Discrimination Kits from Stratagene (La Jolla,Calif.) and various kits from Eurogentec SA (Belgium) and IsogenBioscience BV (The Netherlands).

The oligonucleotide probes and primers of the invention are optionallyprepared using essentially any technique known in the art. In certainembodiments, for example, the oligonucleotide probes and primersdescribed herein are synthesized chemically using essentially anynucleic acid synthesis method, including, e.g., according to the solidphase phosphoramidite triester method described by Beaucage andCaruthers (1981), Tetrahedron Letts. 22(20):1859-1862, which isincorporated by reference, or another synthesis technique known in theart, e.g., using an automated synthesizer, as described inNeedham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168, whichis incorporated by reference. A wide variety of equipment iscommercially available for automated oligonucleotide synthesis.Multi-nucleotide synthesis approaches (e.g., tri-nucleotide synthesis,etc.) are also optionally utilized. Moreover, the primer nucleic acidsdescribed herein optionally include various modifications. To furtherillustrate, primers are also optionally modified to improve thespecificity of amplification reactions as described in, e.g., U.S. Pat.No. 6,001,611, issued Dec. 14, 1999, which is incorporated by reference.Primers and probes can also be synthesized with various othermodifications as described herein or as otherwise known in the art.

In addition, essentially any nucleic acid (and virtually any labelednucleic acid, whether standard or non-standard) can be custom orstandard ordered from any of a variety of commercial sources, such asIntegrated DNA Technologies, the Midland Certified Reagent Company,Eurofins, Biosearch Technologies, Sigma Aldrich and many others.

The term “test sample” as used herein, means a sample taken from anorganism or biological fluid that is suspected of containing orpotentially contains a target sequence. The test sample can be takenfrom any biological source, such as for example, tissue, blood, saliva,sputa, mucus, sweat, urine, urethral swabs, cervical swabs, vaginalswabs, urogenital or anal swabs, conjunctival swabs, ocular lens fluid,cerebral spinal fluid, milk, ascites fluid, synovial fluid, peritonealfluid, amniotic fluid, fermentation broths, cell cultures, chemicalreaction mixtures and the like. The test sample can be used (i) directlyas obtained from the source or (ii) following a pre-treatment to modifythe character of the sample. Thus, the test sample can be pre-treatedprior to use by, for example, preparing plasma or serum from blood,disrupting cells or viral particles, preparing liquids from solidmaterials, diluting viscous fluids, filtering liquids, distillingliquids, concentrating liquids, inactivating interfering components,adding reagents, purifying nucleic acids, and the like.

Advantageously, the invention enables reliable rapid detection ofSARS-CoV-2 in a clinical sample, such as sputum or a nasal or pharyngealswab.

To further illustrate, prior to analyzing the target nucleic acidsdescribed herein, those nucleic acids may be purified or isolated fromsamples that typically include complex mixtures of different components.Cells in collected samples are typically lysed to release the cellcontents, including target nucleic acids. For example, a test samplesuspected of containing virus, can be lysed by contacting viralparticles with various enzymes, chemicals, and/or lysed by otherapproaches known in the art, which degrade, e.g., viral particle walls.In some embodiments, nucleic acids are analyzed directly in the celllysate. In other embodiments, nucleic acids are further purified orextracted from lysates prior to detection. Essentially any nucleic acidextraction methods can be used to purify nucleic acids in the samplesutilized in the methods of the present invention. Exemplary techniquesthat can be used to purifying nucleic acids include, e.g., affinitychromatography, hybridization to probes immobilized on solid supports,liquid-liquid extraction (e.g., phenol-chloroform extraction, etc.),precipitation (e.g., using ethanol, etc.), extraction with filter paper,extraction with micelle-forming reagents (e.g.,cetyl-trimethyl-ammonium-bromide, etc.), binding to immobilizedintercalating dyes (e.g., ethidium bromide, acridine, etc.), adsorptionto silica gel or diatomic earths, adsorption to magnetic glass particlesor organo silane particles under chaotropic conditions, and/or the like.Sample processing is also described in, e.g., U.S. Pat. Nos. 5,155,018,6,383,393, and 5,234,809, which are each incorporated by reference.

A test sample may optionally have been treated and/or purified accordingto any technique known by the skilled person, to improve theamplification efficiency and/or qualitative accuracy and/or quantitativeaccuracy. The sample may thus exclusively, or essentially, consist ofnucleic acid(s), whether obtained by purification, isolation, or bychemical synthesis. Means are available to the skilled person, who wouldlike to isolate or purify nucleic acids, such as DNA, from a testsample, for example to isolate or purify DNA from pharyngeal scrapes(e.g., QIAamp-DNA Mini-Kit; Qiagen, Hilden, Germany).

Equivalents and Scope

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1: Target Selection and Primer Probe Design

Gene sequences for multiple isolates of SARS-CoV-2, closely relatedspecies such as common human coronavirus, severe acute respiratorysyndrome (SARS)-related coronavirus, and Middle East respiratorysyndrome-related coronavirus (MERS-CoV), and for other species commonlyfound in the nasal or pharyngeal samples acquired from patients withupper respiratory symptoms were retrieved from the NCBI database.Sequences were aligned using Clustal omega (Sievers, et al. 2011.Molecular Systems Biology 7:539) and regions with unique specific basesto SARS-CoV-2 species were identified. Loop mediated amplificationprimers were designed using LAMP designer (Premier Biosoft) or anin-house design algorithm. For added specificity, molecular beacons orprobes targeting the amplified products were designed manually or usingBeacon designer (Premier Biosoft). Designed primer sets and beacons werefurther analyzed for specificity using BLAST against the NCBI nucleotidedatabase, including human transcriptome, influenza virus, and non-humancoronavirus. Various primer sets and probes were designed and screenedfor reaction speed.

The inventive primer sets are summarized in Table 3, which include, at aminimum, a forward inner primer (FIP) and backward inner primer (BIP).Additionally, the primer sets typically also include at least twoadditional primers selected from the forward outer primer (F3), backwardouter primer (B3), forward loop primer (LF) and backward loop primer(LB).

TABLE 3 LAMP Primer Sets Set F3 B3 FIP BIP LF LB Set-1  SEQ ID No.: 1 SEQ ID No.: 2  SEQ ID No.: 3  SEQ ID No.: 4  SEQ ID No.: 5  SEQ ID No.:6  Set-2  SEQ ID No.: 7  SEQ ID No.: 8  SEQ ID No.: 9  SEQ ID No.: 10SEQ ID No.: 11 SEQ ID No.: 12 Set-3  SEQ ID No.: 13 SEQ ID No.: 14 SEQID No.: 15 SEQ ID No.: 16 SEQ ID No.: 17 SEQ ID No.: 18 Set-4  SEQ IDNo.: 19 SEQ ID No.: 20 SEQ ID No.: 21 SEQ ID No.: 22 SEQ ID No.: 23 SEQID No.: 24 Set-5  SEQ ID No.: 25 SEQ ID No.: 26 SEQ ID No.: 27 SEQ IDNo.: 28 SEQ ID No.: 29 SEQ ID No.: 30 Set-6  SEQ ID No.: 31 SEQ ID No.:32 SEQ ID No.: 33 SEQ ID No.: 34 SEQ ID No.: 35 SEQ ID No.: 36 Set-7 SEQ ID No.: 37 SEQ ID No.: 38 SEQ ID No.: 39 SEQ ID No.: 40 SEQ ID No.:41 SEQ ID No.: 42 Set-8  SEQ ID No.: 43 SEQ ID No.: 44 SEQ ID No.: 45SEQ ID No.: 46 SEQ ID No.: 47 SEQ ID No.: 48 Set-9  SEQ ID No.: 49 SEQID No.: 50 SEQ ID No.: 51 SEQ ID No.: 52 SEQ ID No.: 53 SEQ ID No.: 54Set-10 SEQ ID No.: 57 SEQ ID No.: 58 SEQ ID No.: 59 SEQ ID No.: 60 SEQID No.: 61 SEQ ID No.: 62 Set-11 SEQ ID No.: 63 SEQ ID No.: 64 SEQ IDNo.: 65 SEQ ID No.: 66 SEQ ID No.: 67 SEQ ID No.: 68 Set-12 SEQ ID No.:69 SEQ ID No.: 70 SEQ ID No.: 71 SEQ ID No.: 72 SEQ ID No.: 73 SEQ IDNo.: 74 Set-13 SEQ ID No.: 63 SEQ ID No.: 86 SEQ ID No.: 65 SEQ ID No.:87 SEQ ID No.: 67 SEQ ID No.: 88 Set-14 SEQ ID No.: 89 SEQ ID No.: 90SEQ ID No.: 91 SEQ ID No.: 92 SEQ ID No.: 93 SEQ ID No.: 94 Set-15 SEQID No.: 89 SEQ ID No.: 96 SEQ ID No.: 97 SEQ ID No.: 98 SEQ ID No.: 99SEQ ID No.: 94 Set-16  SEQ ID No.: 100  SEQ ID No.: 101  SEQ ID No.: 102 SEQ ID No.: 103 SEQ ID No.: 99 SEQ ID No.: 94 Set-17 SEQ ID No.: 89 SEQID No.: 96 SEQ ID No.: 91 SEQ ID No.: 92 SEQ ID No.: 93 SEQ ID No.: 94

Typically, 3 to 5 μL of RNA standards or genomic RNA or genomic RNAextracted from negative nasal swab matrix or negative controls(NTC=nuclease free water or Tris buffer, no template control) served astemplate for RTLAMP reactions. 10-25 μl total volume reactions wereprepared on ice as mixes containing formulations including 1×amplification buffer comprising 10-40 mM Tris-HCl, 0-0.5% Tween 20,0-300 mM Trehalose, 5-70 mM KCl, 4-41 mM MgSO₄, 10-20 mM (NH₄)₂SO₄, 0-2mM TCEP and 1.6-2 mM each dCTP, dGTP, dATP and dTTP. NEB Bst2 polymerase(NEB CN #M0537L) and RTx Warmstart reverse transcriptase (NEB CN#M0380S) enzymes. Primers (2 μM inner primers, 0.2 μM outer primers, and0.8 μM Loop primers) were added to individual reactions or directly tomaster mixes as required per experimental design. Molecular beacons (0.2μM) or 200 nM To-Pro dye was also added to the master mix, as indicatedin the examples below. Amplification reactions were prepared with thestandard 6-primer composition. Master mixes were distributed toindividual sample templates, vortexed and centrifuged briefly and eachreaction loaded into individual wells of a 96 or 384 well plate (RocheCN #4729692001 or BioRad CNhsI9605). Reactions were carried out attemperatures ranging from 60-67° C. and fluorescence monitored on eithera Roche LightCycler 96 Real-Time PCR instrument or a BioRad CFX96 realtime cycler. Target amplification was monitored via intercalating dye ormolecular beacon probe binding to target resulting in release ofmolecular beacon fluorescence intramolecular quenching.

Example 2: Amplification Reaction Kinetics

Input samples were RNA molecular standards generated from in vitrotranscription of 900-1500 bp gene fragments, including those ofSARS-CoV-2 and common human coronavirus.

An RNA molecular standard or genomic RNA was diluted to variousconcentrations ranging from 50-5000 copies/reaction to assess thesensitivity of the indicated primer set (Table 3). Standards wereserially diluted with 0.1 mg/mL Poly A carrier RNA in PBS (Sigma) andused as template for amplification in RTLAMP reactions. ToPro™ or Syto™dye or a compatible wavelength version within the same dye set family(Life Technologies; green or red fluorescent carbocyanine nucleic acidstain) was used for the detection of the amplified product. The mastermix was prepared as described in Example 1.

This example shows that using this set of primers and the loop mediatedamplification method, fast amplification kinetics are achieved. Resultsare summarized in Table 4, in which the Time to Positive (T_(p)) wascalculated by using an in-house developed algorithm. NT indicatesconcentrations not tested. Results are classified by the time topositive (NT means “not tested” and “no call” indicates that noamplification was detected).

TABLE 4 Time to Positive Dye Detection Time to Positive (minutes) PrimerSet 5,000/reaction 500/reaction 50/reaction NTC Set-1 7.8 9.1 13.0 37.2Set-2 5.8 6.6 8.4 no call Set-3 NT 8.2 9.2 no call Set-4 8.6 9.7 13.3 nocall Set-5  8.54 10.0  no call no call Set-6 6.8 8.0 no call no callSet-7 7.7 8.8 12.4 no call Set-8 6.7 8.3 10.5 24.0 Set-9 6.5 7.8 9.6 nocall  Set-10 5.6 6.5 9.6 28.3  Set-11 4.8 5.3 6.5 no call  Set-12 7.79.0 11.9 no call  Set-14 8.5 9.7 11 no call

A negative nasal swab matrix was spiked with genomic material fromcommon human coronaviruses strain 229E, NL63, and OC43. Thecorresponding extracted nucleic acids or DNAs were used as templates inRT-LAMP reactions containing the LAMP primers and compared to 500-5000copies/reaction of SARS-COV-2 RNA standard for specificity.

TABLE 5 Cross-Reactivity Dye Detection Primers 5000 500 229E NL63 OC43Set-1 7 8.5 no call no call no call Set-4 7 7.9 no call no call no callSet-8 6.3 7.2 no call no call no call  Set-11 4.8 5.3 no call no call nocall

Example 3: Molecular Beacon Detection

To provide an additional level of direct sequence based detection ofamplified product (as opposed to indirect dye detection), molecularbeacons (MB1-MB14; see Table 2) targeting unique nucleotides within theSARS-CoV-2 amplicon of primer sets with promising times-to-positivecombined with sensitivity, were designed and utilized for detection ofamplification from RNA standards (Table 4). The molecular beacon probewas designed with 5′ fluorophore/3′ quencher modifications(6-Carboxyfluorescein (FAM)) and Black Hole Quencher 1 (BHQ1) and LockedNucleic Acid (+) included to provide target-specific fluorescentdetection.

10-25 μL total volume reactions were evaluated utilizing 25 to 500copies/reaction of SARS-CoV-2 RNA standard or genomic RNA as templateinput according to the methods described in Examples 1 and 2. While useof a Molecular Beacon for detection resulted in a slight increase inreaction Tp, the ability to directly detect amplification products basedon sequence, and thereby distinguish closely related species, provides areasonable tradeoff.

TABLE 6 Time to Positive Probe Detection Time to Positive (minutes)Primers Beacon 500 50 25 NTC Set-1 MB1 10.1 14.7 13.9 no call Set-8 MB29.1 12.0 12.4 no call Set-9 MB4 9.4 14.9 14.5 no call Set-9 MB8 8.1 12.7NT no call Set-9 MB9 8.2 10.6 NT no call Set-9  MB10 9.2 11.6 NT no call Set-11 MB5 7.1 8.3 8.9 no call  Set-11 MB7 NT 7.8 8.5 no call  Set-12MB6 11.3 13.7 14.7 no call Set-9  MB11 7.1 8.5 12.6 no call  Set-14 MB14 NT 13.7 13.7 no call

Selected primer set and Molecular Beacon pair was additionally testedfor specificity by comparing reactions with 500 to 5000 copies/reactionof SARS-CoV-2 RNA standard to reactions with approximately 5×10⁵copies/reaction of RNA standards or extracted nucleic acids fromnegative nasal swab matrix spiked with closely related common humancoronavirus strain 229E, NL63, OC43, or SARS-related coronavirus. Whenthe amplification reactions were performed as described in Example 1 and2, the primer and Molecular Beacon pair tested had no cross-reactivityagainst common human coronavirus (Table 7).

TABLE 7 Cross Reactivity Probe Detection Primers Beacon 5000 500 229ENL63 OC43 SARS Set-8  MB2 8.4 9.1 no call no call no call NT Set-10 MB36.3 7.1 NT NT NT no call Set-12 MB6 10.1  11.3  NT NT NT no call

Example 4: Multi-Target Amplification

The genomic sequence of SARS-CoV-2, like all viruses, is subject tomutation and natural selection. Given the potential lethality andrealtively high transimissability of SARS-CoV-2, it would be beneficialfor an assay to detect the presence of more than one genomic target.Thus, if one portion of the virus has mutated, the virus can still bedetecte via a second location. This can be achieved by performing twoseparate amplification assays. However, time, personnel and resourcesare limited. Accordingly, it is advantageous if two or more genomictargets can be assessed simultaneously in the same reaction well. Thisis commonly performed with standard PCR assays, which typically utilizetwo primers per amplification locus. LAMP, however, typically includessix primers comprising eight separate hybridization sites. This createssignificantly higher potential for primer:primer interactions that couldinterfere with targeted amplification or lead to off-targetamplification that could be confused for on-target amplification(leading to a mistaken positive diagnosis for COVID-19). Set-9 andSet-11 were confirmed in silico to present little potential foroff-target amplication from primer-primer interactions.

First, Set-9 and Set-11 were combined to amplify an RNA molecularstandard, diluted to 25, 50, or 500 copies per reaction variousconcentrations ranging from 5-500 copies/reaction to assess thesensitivity combined primer set. Standards were serially diluted with0.1 mg/mL Poly A carrier RNA in PBS (Sigma) and used as template foramplification in RTLAMP reactions. ToPro™ or Syto™ dye was used for thedetection of the amplified product. The master mix was prepared asdescribed in Example 1. Amplification was detected at 5.3 minutes (timeto positive) for 500 copies/reaction; 6.2 minutes for 50 copies/reactionand 6.5 minutes for 25 copies/reaction. No amplication was detectionwith the negative control, as expected.

A negative nasal swab matrix was spiked with genomic material fromcommon human coronaviruses strain 229E, NL63, and OC43. Thecorresponding extracted nucleic acids or DNAs were used as templates inRT-LAMP reactions containing combined LAMP primer set and compared to50-5000 copies/reaction of SARS-COV-2 RNA standard or extracted genomicRNA from negative nasal swab matrix for specificity. No amplificationwas detected for any potentially cross-reacting species.

To provide an additional level of direct sequence-based detection ofamplified product (as opposed to indirect dye detection), molecularbeacons (MB5 and MB11; See Table 2) targeting unique nucleotides withinthe SARS-CoV-2 ORF1b and N gene amplicons, respectively, were includedto provide target-specific fluorescent detection. 10-25 μL total volumereactions were evaluated utilizing 25, 50 or 500 copies/reaction ofSARS-CoV-2 RNA standard as template input according to the methodsdescribed in Examples 1 and 2. The combined primer set (Set-9 andSet-11) plus two beacons (MB5 and MB11) detected application at 6.5minutes (time to positive) at 500 copies/reaction; at 7.7 minutes at 50copies/reaction and at 7.8 minutes at 25 copies/reaction. Noamplification was detected with a no template (negative) control.

The combined primer set and Molecular Beacon pair was additionallytested for specificity by comparing reactions with 50 to 5000copies/reaction of SARS-CoV-2 RNA standard or extracted genomic RNA fromnegative nasal swab matrix to reactions with approximately 5×10⁵copies/reaction of RNA standards or extracted nucleic acids fromnegative nasal swab matrix spiked with closely related common humancoronavirus strain 229E, NL63, OC43, or SARS-related coronavirus. Whenthe amplification reactions were performed as described in Example 1 and2, the primer (Set-9 and Set-11, together) and Molecular Beacon pair(MB5 and MB 11, together) detected no cross-reactivity against commonhuman coronavirus 229E, human coronavirus NL83, human coronavirus OC43,and SARS-CoV (2003).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1.-30. (canceled)
 31. A composition comprising SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO:
 54. 32.The composition of claim 31, further comprising a molecular beaconcomprising a fluorophore, a quencher and a polynucleotide.
 33. Thecomposition of claim 32, wherein the polynucleotide comprises a sequenceselected from the group consisting of: nucleotides 5-22 of SEQ ID NO:75, nucleotides 5-22 of SEQ ID NO: 76, nucleotides 5-22 of SEQ ID NO:80, nucleotides 5-22 of SEQ ID NO: 81, nucleotides 4-22 of SEQ ID NO:82, nucleotides 6-28 of SEQ ID NO: 83, nucleotides 6-25 of SEQ ID NO:84, and nucleotides 3-23 of SEQ ID NO:
 85. 34. The composition of claim33, wherein the polynucleotide comprises a sequence selected from thegroup consisting of: SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 80, SEQ IDNO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85.35. The composition of claim 34, wherein the polynucleotide consists asequence selected from the group consisting of: SEQ ID NO: 75, SEQ IDNO: 76, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQID NO: 84, and SEQ ID NO:
 85. 36. The composition of claim 32, whereinthe fluorophore is FAM and the quencher is BHQ1.
 37. The composition ofclaim 32, wherein the fluorophore is ATTO 565 or Alexa 594 and thequencher is BHQ1 or BHQ2.
 38. The composition of claim 31, furthercomprising an intercalating dye.
 39. The composition of claim 31,further comprising a strand displacement DNA polymerase and a reversetranscriptase.
 40. A composition comprising a set of polynucleotidesselected from the group consisting of: Set-5 and Set-9.
 41. A method ofdetecting SARS-CoV-2 in a test sample, the method comprising: (a)extracting nucleic acid from the test sample; (b) amplifying a targetsequence by reacting the nucleic acid extracted in step (a) with areaction mixture comprising a strand displacement DNA polymeraseactivity, a reverse transcriptase activity, and a sequence-specificprimer set, wherein said sequence-specific primer set is selected fromthe group consisting of: Set-5 and Set-9; and (c) detecting the presenceor absence of an amplification product from step (b); wherein thepresence of said amplification product is indicative of the presence ofSARS-CoV-2 in the test sample.
 42. The method of claim 41, wherein theamplification in step (b) of the target sequence is performed betweenabout 60° C. and about 67° C. for less than 30 minutes.
 43. The methodof claim 42, wherein the amplification step is performed for less thanfifteen minutes.
 44. The method of claim 43, wherein the amplificationstep is performed for less than twelve minutes.
 45. The method of claim44, wherein the amplification step is performed for less than nineminutes.
 46. The method of claim 41, wherein detecting the presence orabsence of the amplification product comprises hybridizing theamplification product with a probe comprising a polynucleotide attachedto a label.
 47. The method of claim 46, wherein the label is afluorophore.
 48. The method of claim 47, wherein the fluorophore iscovalently attached to a terminus of the polynucleotide.
 49. The methodof claim 46, wherein the labeled polynucleotide comprises a sequenceselected from the group consisting: of nucleotides 5-22 of SEQ ID NO:75, nucleotides 5-22 of SEQ ID NO: 76, nucleotides 5-22 of SEQ ID NO:80, nucleotides 5-22 of SEQ ID NO: 81, nucleotides 4-22 of SEQ ID NO:82, nucleotides 6-28 of SEQ ID NO: 83, nucleotides 6-25 of SEQ ID NO:84, and nucleotides 3-23 of SEQ ID NO:
 85. 50. The method of claim 49,wherein the labeled polynucleotide comprises a sequence selected fromthe group consisting of: SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 80,SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, and SEQ IDNO:
 85. 51. The method of claim 50, wherein the sequence-specific primerset is Set-9 and the sequence of the labeled polynucleotide is SEQ IDNO:
 83. 52. A kit comprising the composition of claim
 31. 53. The kit ofclaim 52, further comprising a strand displacement polymerase and areverse transcriptase.
 54. The kit of claim 53, further comprising amolecular beacon comprising a fluorophore, a quencher, and apolynucleotide.
 55. The kit of claim 54, wherein the polynucleotidecomprises a sequence selected from the group consisting of: nucleotides5-22 of SEQ ID NO: 75, nucleotides 5-22 of SEQ ID NO: 76, nucleotides5-22 of SEQ ID NO: 80, nucleotides 5-22 of SEQ ID NO: 81, nucleotides4-22 of SEQ ID NO: 82, nucleotides 6-28 of SEQ ID NO: 83, nucleotides6-25 of SEQ ID NO: 84, and nucleotides 3-23 of SEQ ID NO:
 85. 56. Thekit of claim 55, wherein the polynucleotide comprises a sequenceselected from the group consisting of: SEQ ID NO: 75, SEQ ID NO: 76, SEQID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,and SEQ ID NO:
 85. 57. The kit of claim 56, wherein the polynucleotideconsists a sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ IDNO: 83, SEQ ID NO: 84, and SEQ ID NO:
 85. 58. The kit of claim 57,wherein the polynucleotide consists of SEQ ID NO: 83.